In this work we propose to advance the state of the art in two-photon, three-dimensional molecular tracking and create a unique microscopy system to explore open questions in subcellular trafficking and spatial distribution of receptor tyrosine kinases (RTKs) in cancer cells. Mounting evidence indicates that EGFR (epidermal growth factor receptor, an RTK) can be shuttled from the cell surface to a variety of cellular organelles, including Golgi apparatus, ER, mitochondria, and nucleus. Clearly, EGFR serves specific biological functions while residing inside these organelles, but we have very limited knowledge of EGFR's translocation routes and the associated subcellular signaling pathways. This is mainly due to the fact that we lack proper tools that can clearly map EGFR trafficking trajectories to cellular compartments and organelles in 3D space and observe protein-protein interactions along the transport route in real time. Here we propose to integrate a uniquely designed 3D molecular tracking system with the STED (stimulated emission depletion) super-resolution imaging technique to achieve real-time, superimposed molecular trajectories of EGFRs on super-resolution images of organelles. Moreover, with protein-protein interaction analysis capability, we will be able to study EGFR trafficking accompanied by the associated proteins in real time. Information acquired from our innovative system will shed light on both receptor biology and potential therapeutic targets of anti-EGFR therapies for clinical applications. As a strategic goal for NIH, personalized medicine currently emphasizes on molecular signatures such as genetic, epigenetic and proteomic variations of individual tumors. Variations in the subcellular trafficking and distribution of RTKs within cancer cells coul also serve as an additional molecular signature for therapeutic choice, as those variations could have a dramatic impact on therapeutic efficacy.